Presently, if one wants to accurately assess the concentrations of chemicals or drugs inside a living animal, a sample of the blood or tissue to be studied is removed from the animal and taken to an analytical laboratory to have the chemicals of interest extracted and quantified. Typically, a first step is a pre-treatment of the sample to convert it to a form more suitable for chemical extraction. In the case of blood, this may be by the removal of blood cells and/or some blood components by the preparation of serum or plasma. In the case of a tissue sample, this may be by many processes, including: freezing, grinding, homogenizing, enzyme treatment (e.g., protease or cellulase) or hydrolysis. Subsequently, compounds of interest are extracted and concentrated from the processed sample. For example serum samples may be subjected to liquid-liquid extraction, solid phase extraction or protein precipitation, followed by drying and reconstitution in an injection solvent. A portion of the injection solvent is introduced to an analytical instrument for chromatographic separation and quantification of the components. This method produces accurate results with high specificity for the compound of interest, but is time consuming and labor intensive. Also, because of the large number of steps in the process there is a significant chance of errors in sample preparation impacting the results. This method has good sensitivity and selectivity and accuracy for the target compounds but is limited in that the chemical balance inside the animal is disrupted during sampling. In many cases, this disruption reduces the value of the results obtained, and in some cases makes this technique inappropriate for the analysis. Where the removed blood volume is a high proportion of the total blood volume of the animal, as is commonly the case when mice are used, the death of the animal results. This means that a different animal must be used for each data point and each repeat By eliminating the need for a blood draw in this case, fewer animals would be required for testing and a significant improvement in inter-animal variation in the results would be achieved.
Alternatively, biosensors have been developed for some applications of analysis of chemical concentrations inside animals. In this case, a device consisting of a specific sensing element with an associated transducer is implanted. The device produces a signal collected by an electronic data logger that is proportional to the chemicals to which the sensor responds. The main limitations of this type of device are that they normally respond to a spectrum of chemicals rather than having specificity for only one chemical. Of the spectrum of chemicals to which the sensor responds, some produce a greater and some a lesser response. Sensors are also susceptible to interferences where another chemical present in a system interferes with the response produced by the target chemicals. It is for these reasons that biosensors are normally limited in terms of accuracy and precision. Additionally, biosensors are typically not as sensitive to low chemical concentrations as state-of-the-art, stand-alone, detectors. Such detectors, for example mass spectrometers, are used in the above mentioned conventional analysis techniques and in solid phase microextraction.
Microextraction is a significant departure from conventional ‘sampling’ techniques, where a portion of the system under study is removed from its natural environment and the compounds of interest extracted and analyzed in a laboratory environment. As with any microextraction, compounds of interest are not exhaustively removed from the investigated system, and conditions can be devised where only a small proportion of the total amount of compound, and none of the matrix, are removed. This avoids disturbing the normal balance of chemical components. This could have a benefit in the non-destructive analysis of very small tissue sites or samples. Because extracted chemicals can be separated chromatographically and quantified by highly sensitive analytical instruments, high accuracy, sensitivity and selectivity are achieved.
With current commercially available solid phase microextraction (SPME) devices, a stationary extraction phase is coated onto a fused silica fiber. The coated portion of the fiber is typically about 1 cm long and coatings have various thicknesses. The fiber can be mounted into a stainless steel support tube and housed in a syringe-like device for ease of use. Extractions are performed by exposing the extraction phase to a sample for a we-determined time to allow sample components to come into equilibrium with the extraction phase. After extraction, the fiber is removed to an analytical instrument (typically a gas or liquid chromatograph) where extracted components are desorbed and analysed. The amount of a component extracted is proportional to its concentration in the sample (J. Pawliszyn “Method and Device for Solid Phase Microextraction and Desorption”, U.S. Pat. No. 5,691,206).
To date, commercial SPME devices have been used in some applications of direct analysis of living systems. For example they have been applied for the analysis of airborne pheromones and semiochemieals used in chemical communications by insects (Moneti, Dani, F. R.; Pieraccini, G. T. S. Rapid Commun. Mass Spectrom. 1997, 11, 857-862), (Frerot, B.; Malosse, C.; Cain, A. H. J. High Resolut. Chromatogr, 1997, 20, 340-342) and frogs (Smith, B. P.; Zini, C. A.; Pawliszyn, J.; Tyler, M. J.; Hayasaka, Y.; Williams, B.; Caramao, E. R. Chemistry and Ecology 2000, 17, 215-225) respectively. In these cases, the living animals were non-invasively monitored over time by assessing the chemical concentrations in the air around the animal, providing a convenient means to study complicated dynamic processes without interference.
The current commercial devices do, however, have some limitations for in vivo and in vitro analysis of a biological matrix, such as blood or tissue. Firstly, the most difficult and undesirable problem is the adsorption of proteins and other macromolecules on the surface of SPME fibers. Macromolecules are understood to be biological components with a molecular mass greater than about 10,000 atomic mass units. These macromolecules constitute a diffusion barrier and decrease the extraction efficiency in subsequent experiments. In order to transfer all SPME advantages to the field of in vivo and in vitro analysis of biological samples, it is imperative to develop new biocompatible devices suitable for extracting compounds from biological matrices.
Devices can be made biocompatible by coating them with a biocompatible material. Custom-made coatings based on polypyrrole (PPY) (Lord, H. L.; Grant, R. P.; Walles, M.; Incledon, B.; Fahie, B.; Pawliszyn, J. B., Anal. Chem. 2003, 75(19), 5103-5115) and poly(ethylene glycol) (PEG) (Musteata, F. M.; Musteata, M. L; Pawliszyn, J., Clin Chem 2006, 52(4), 708-715) have been used for in vivo drug analysis. Other biocompatible materials include restricted access materials (RAM, ionic liquids (IL), polydimethylsiloxane (PDMS), polypyrrole, and polyethylene glycol). Biocompatible membranes have also been prepared from polyacrylonitrile (Nie, F.-Q.; Xu, Z.-K.; Ming, Y.-Q.; Kou, R.-Q.; Liu, Z.-M.; Wang, S.-Y. Desalination 2004, 160, 43-50. Lavaud, S.; Canivet, E.; Wuillai, A.; Maheut, H.; Randoux, C.; Bonnet, J.-M.; Renaux, J.-L.; Chanard, J. Nephrology, Dialysis, Transplantation 2003, 18, 2097-2104. Yang, M. C.; Lin, W. C. Journal of Polymer Research 2002, 9, 201-206), polyurethane, chitosan, and cellulose.
Polymers such as polypyrroles, derivatised cellulose, polysulfones, polyacrylonitrile (PAN), polyethylene glycol and polyamides are currently used to prepare biocompatible membranes used for separation of sub-micron particles in biomedical applications. PAN has been widely used as membrane material in the fields of dialysis and ultrafiltration. It has been found that its properties can be fine-tuned by using specific co-monomers. The terms “polyacrylonitrile” and “PAN” are used herein to refer to homopolymers as well as copolymers of acrylonitirile containing at least about 85% by weight acrylonitrile and up to about 15% by weight of at least one other ethylenically unsaturated compound copolymerizable with acrylonitrile. For example, PAN can be tailored with a reactive group for enzyme immobilization. Furthermore, some co-monomers lead to improved mechanical strength, solvent resistance, high permeation flux, and biocompatibility. Accordingly, PAN-based membranes have great potential for the treatment of wastewater, the production of ultra-pure water, hemodialysis artificial kidneys, and biocatalysis with separation. PAN is one of the most important polymers used in the biomedical area because of its exceptional qualities, such as good thermal, chemical, and mechanical stability as well as biocompatibility. Membranes made of PAN are widely used as dialyzers able to remove low to middle molecular weight proteins and for high-flux dialysis therapy. PAN is one of the best polymers in terms of biocompatibility.
However, good extractive materials are generally not biocompatible and PAN is not appropriate as an extractive material for SPME.
It is, therefore, desirable to provide a biocompatible composition able to extract small molecules from a matrix for use with solid phase microextraction devices, as well as a process for coating SPME fibers with said composition.